1. Field of the Invention
The present invention relates generally to a method for resolving mixtures of chemical compounds by a chromatographic process. More particularly, the method of the present invention is directed to a particular mobile phase for use in reversed phase high performance liquid chromatography for use in resolving mixtures of chemical compounds, particularly peptides.
Chromatography is a separation method whereby individual chemical compounds which were originally present in a mixture are resolved from each other by the selective process of distribution between two phases. The distribution of chemical species to be separated occurs in a dynamic process between a mobile phase and a stationary phase. In liquid-liquid chromatography, a liquid stationary phase is held on the surface of an inert solid which serves as its support and, ideally, does not participate in the separation process. The components of a mixture having different solubilities in the stationary phase separate by migrating at different rates.
In modern liquid chromatography, the sample (a mixture of components to be separated) is introduced into a controlled stream of mobile phase at the top of the column. The effluent from the column is continuously monitored by a suitable detector, and the signals are recorded or otherwise processed. The stream of mobile phase is usually pumped under conditions of high pressure, i.e., pressure in the range of 500 to 6000 psig. Modern liquid chromatography utilizing a controlled stream of mobile phase under high pressure is usually referred to as high performance liquid chromatography (HPLC).
In liquid-liquid chromatography, the mobile phase and the stationary phase must be immiscible. Since immiscibility means automatically that the two phases must have significantly different chemical properties, only combinations of either a nonpolar mobile phase and a polar stationary phase or a polar mobile phase and a nonpolar stationary phase are feasible. Through convention, a system that uses a nonpolar stationary phase and a relatively polar mobile phase is referred to as reversed-phase chromatography. Although there is no justification to consider any stationary phase "normal," the term "reversed-phase" has persisted as a convenient general description for nonpolar stationary phases.
2. Background of the Prior Art
The mobile phase composition is an important variable in liquid-liquid chromatography. It is a particularly important variable in resolving mixtures of peptides which can have as few as two amino acids and as many as several hundred. Separation of optically different peptides, such as the dipeptide pairs L-Phe-L-Leu and L-Phe-D-Leu, are a further problem in resolving mixtures of peptides. Various mobile phase compositions are known for use in resolving peptide mixtures, such as acetonitrile in combination with a mineral salt of phosphoric acid. Such known mobile phase compositions have shown good resolution and usefulness in resolving lower molecular weight peptides, but poor resolution and recovery has been encountered when dealing with polypeptides, such as .beta.-endorphins and proteins. A further problem encountered with use of acetonitrile and the mineral salt of phosphoric acid mobile phase compositions is that such mobile phase compositions are detrimental to the column when resolving mixtures of higher molecular weight peptides, such as human .beta.-endorphin, ovine .beta.-endorphin and porcine .beta.-endorphin.
It would be desirable to provide a mobile phase composition which has high resolution and good recovery in respect to a broad range of peptides and other chemical compounds and which has high selectivity in respect to minor chemical and optical differences between closely related peptides.